Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
The materials, instruments and reagents used in the invention are all commercially available unless otherwise specified, and the experimental methods used are all routine in the art unless otherwise specified.
The nucleotide sequence of the loop RNAhsa _circ_0096498 (abbreviated as circ 96498) in the following examples is shown in SEQ ID NO: 1:
AGGAACAGAAAGAAAGTGAAAAGATGAAAAGTGAGGAGCAGCCTATGGATTTAGAAAACCGTTCTACAGCCAATGTTCTAGAAGAGACTACTGTGAAAAAAGAAAAAGAAGATGAAAAGGAACTTGTGAAACTGCCAGTCATAGTGAAGCTAGAAAAACCTTTGCCAGAAAATGAAGAAAAAAAGATTATCAAAGAAGAAAGTGATTCCTTCAAGGAAAATGTCAAACCCATTAAAGTTGAGGTGAAGGAATGTAGAGCAGATCCTAAAGATACCAAAAGTAGCATGGAGAAGCCAGTGGCACAGGAGCCTGAAAGGATCGAATTTGGTGGCAATATTAAATCTTCTCACGAAATTACTGAGAAATCTACTGAAGAAACTGAGAAACTTAAAAATGACCAGCAGGCCAAGATACCACTAAAAAAACGAGAAATTAAACTGAGTGATGATTTTGACAGTCCAGTCAAGGGACCTTTGTGTAAATCAGTTACTCCAACAAAAGAGTTTTTGAAAGATGAAATAAAACAAGAGGAAGAGACTTGTAAAAGGATCTCTACAATCACTGCTTTGGGTCATGAAGGGAAACAGCTGGTAAATGGAGAAGTTAGTGATGAAAGGGTAGCTCCAAATTTTAAGACAGAACCAATAGAGACAAAGTTTTATGAGACAAAGGAAGAGAGCTATAGCCCCTCTAAGGACAGAAATATCATCACGGAGGGAAATGGAACAGAGTCCTTAAATTCTGTCATAACAAGTATGAAAACAGGTGAGCTTGAGAAAGAAACAGCCCCTTTGAGGAAAGATGCAGATAGTTCAATATCAGTCTTAGAGATCCATAGTCAAAAAGCACAAATAGAGGAACCCGATCCTCCAGAAATGGAAACTTCTCTTGATTCTTCTGAGATGGCAAAAGATCTCTCTTCAAAAACTGCTTTATCTTCCACCGAGTCGTGTACCATGAAAGGTGAAGAGAAGTCTCCCAAAACTAAGAAGGATAAGCGCCCACCAATCCTAGAATGTCTTGAAAAGTTAGAGAAGTCCAAAAAGACTTTTCTTGATAAGGACGCACAAAGATTGAGTCCAATACCAGAAGAAGTTCCAAAGAGTACTCTAGAGTCAGAAAAGCCTGGCTCTCCTGAGGCAGCTGAAACTTCTCCACCATCTAATATCATTGACCACTGTGAGAAACTAGCCTCAGAAAAAGAAGTGGTAGAATGCCAGAGTACAAGTACTGTTGGTGGCCAGTCTGTGAAAAAAGTAGACCTAGAAACCCTAAAAGAGGATTCTGAGTTCACAAAGGTAGAAATGGATAATCTGGACAATGCCCAGACCTCTGGCATAGAGGAGCCTTCTGAGACAAAGGGTTCTATGCAAAAAAGCAAATTCAAATATAAGTTGGTTCCTGAAGAAGAAACCACTGCCTCAGAAAATACAGAGATAACCTCTGAAAGGCAGAAAGAGGGCATCAAATTAACAATCAGGATATCAAGTCGGAAAAAGAAGCCCGATTCTCCCCCCAAAGTTCTAGAACCAGAAAACAAGCAAGAGAAGACAGAAAAGGAAGAGGAGAAAACAAATGTGGGTCGTACTTTAAGAAGATCTCCAAGAATATCTAGACCCACTGCAAAAGTGGCTGAGATCAGAGATCAGAAAGCTGATAAAAAAAGAGGGGAAGGAGAAGATGAGGTGGAAGAAGAGTCAACAGCTTTGCAAAAAACTGACAAAAAGGAAATTTTGAAAAAATCAGAGAAAGATACAAATTCTAAAGTAAGCAAGGTAAAACCCAAAGGCAAAGTTCGATGGACTGGTTCTCGGACACGTGGCAGATGGAAATATTCCAGCAATGATGAAAGTGAAGGGTCTGGCAGTGAAAAATCATCTGCAGCTTCAGAAGAGGAGGAAGAAAAGGAAAGTGAAGAAGCCATCCTAGCAGATGATGATGAACCATGCAAAAAATGTGGCCTTCCAAACCATCCTGAGCTAATTCTTCTGTGTGACTCTTGCGATAGTGGATACCATACTGCCTGCCTTCGCCCTCCTCTGATGATCATCCCAGATGGAGAATGGTTCTGCCCACCTTGCCAACATAAACTGCTCTGTGAAAAATTAGAGGAACAGTTGCAGGATTTGGATGTTGCCTTAAAGAAGAAAGAGCGTGCCGAACGAAG(SEQ ID NO：1).
example 1 radiation induces up-regulation of hepatic stellate cell circ96498 expression.
The pre-radiotherapy cancer tissues of 20 cases 2018-2022 of hepatocellular carcinoma patients who were subjected to radiotherapy in southern hospitals of southern medical university were collected, the total radiotherapy dose was 40-60Gy, the number of divisions was 5-20, all patients were subjected to CT and MRI examination before and after radiotherapy, and the Non-radioactive liver injury group (Non-RILI group, n=10) and the radioactive liver injury group (RILI group, n=10) were classified according to whether or not the radioactive liver injury occurred by MRI imaging. The expression of circ96498 and α -SMA in both groups of samples was examined using Fluorescence In Situ Hybridization (FISH), and the co-localization of circ96498 with hepatic stellate cells was assessed, showing that the expression of both circ96498 and α -SMA in the RILI group was significantly up-regulated and that there was better co-localization of both, indicating that circ96498 was significantly elevated in RILI patient expression and specifically expressed in hepatic stellate cells (fig. 1 a).
The expression of circ96498 in LX2 was examined by qRT-PCR after 24h irradiation of human hepatic stellate cells LX2 with different doses of radiation 0, 2, 4, 6, 8, 10Gy, respectively, and as a result, it was found that circ96498 was up-regulated after LX2 irradiation, and that 8Gy up-regulation was most pronounced, indicating that the irradiation could induce up-regulation of hepatic stellate cell circ96498 (B in fig. 1). In addition, agarose gel electrophoresis and Sanger sequencing also confirmed the cyclization characteristics of circ96498 (C, D in FIG. 1).
The detection method of fluorescence in situ hybridization and qRT-PCR is as follows:
(1) Fluorescence in situ hybridization
① Slicing and baking, namely fixing the collected specimens for 48 hours by using 4% paraformaldehyde, and dehydrating tissues and embedding paraffin. Slicing, and placing the slices in a 65 ℃ oven for 2 hours before an experiment;
② Dewaxing and alcohol gradient dehydration, namely, xylene I10min, xylene II 10min, xylene III 10min, 100% ethanol I5 min, 100% ethanol II 5min, 90% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min and PBS soaking for 5min;
③ High pressure antigen retrieval, namely pouring the prepared 1 Xcitric acid buffer solution into a box, overflowing the box through a glass slide, putting the box into a pressure cooker, and adding a small amount of citric acid buffer solution into the pressure cooker. When the water is boiled, the cover is covered, and after the air is sprayed for 5min, the heating is stopped. Taking out the wafer box and cooling at room temperature;
④ Proteinase K treatment, namely, removing redundant liquid on tissues, dripping a proper amount of proteinase K working solution on the tissues, and standing at 37 ℃ for 25min. Shaking and washing with PBS for 3 times at 100rpm/min for 5min each time;
⑤ And (3) pre-hybridization working solution treatment, namely throwing away redundant liquid on the slice, dripping the pre-hybridization working solution to cover tissues, incubating for 2 hours in a 42 ℃ wet box, and covering a cover slip special for in situ hybridization. Shaking and washing with PBS for 3 times at 100rpm/min for 5min each time;
⑥ Hybridization 1, namely removing redundant liquid on the slice, dripping hybridization working solution, wherein the probe concentration is 1:5 (the probe is diluted by salmon sperm DNA super hybridization solution containing 100 ug/ml), covering an in-situ hybridization glass slide, hybridizing at 42 ℃ overnight, removing a cover slip, and shaking and washing 3 times by PBS (phosphate buffer solution) at 100rpm/min for 5min each time;
⑦ Hybridization 2, spin-drying slice gently, dripping preheated probe mixture 2 hybridization solution (60 μl), horizontally placing in a wet box, hybridizing at 40deg.C for 45min, shaking with PBS for 3 times, 100rpm/min, and 5min each time;
⑧ Signal hybridization, namely lightly spin-drying slices, dripping preheated signal probe hybridization solution (60 mu L), horizontally placing in a wet box, hybridizing for 45min at 40 ℃, and shaking and washing for 3 times by PBS (phosphate buffer solution) at 100rpm/min for 5min each time;
⑨ DAPI counterstaining, namely dripping DAPI dye solution into the slice, and incubating for 5min in dark. The mixture was shaken 3 times with PBS at 100rpm/min for 5min each time. After flushing, dripping anti-fluorescence quenching sealing tablet sealing sheets;
⑩ And (3) microscopic photographing, namely observing and collecting images under a Nikon normal fluorescence microscope.
(2)qRT-PCR
① Cell total RNA was extracted 1) the supernatant was discarded, and after washing 1 time with PBS, 1mL of Trizol reagent was added to the cells of the 6-well plate, and the mixture was allowed to stand on ice for 5 minutes, followed by collection into a 1.5mL EP tube. 2) 200 mu L of chloroform (1/5 Trizol reagent) is added into the EP tube, the EP tube is turned upside down for 5-6 times, the EP tube is fully and evenly mixed, the EP tube is kept stand for 5min at room temperature, after the liquid in the EP tube is layered, the EP tube is placed into a 4 ℃ centrifuge for centrifugation at 12000rpm/min, and the EP tube is centrifuged for 15min. 3) After centrifugation, the mixture was seen to separate into an upper colorless aqueous phase containing total RNA and a lower pale red phenol-chloroform phase. Collecting upper layer liquid in the EP pipe to a new EP pipe, adding equal volume of isopropanol, turning the EP pipe up and down for 5-6 times, and standing at room temperature for 10min. Then centrifuged at 4℃at 12000rpm/min for 10min. 4) The supernatant was removed, 1mL of 75% ethanol (prepared with DEPC water) was added to the tube to wash the RNA pellet, and the pellet was placed in a 4℃centrifuge at 12000rpm/min for 5min. This step was repeated 2 times. 5) The supernatant was discarded, dried thoroughly in an ultra clean bench, and finally dissolved in 20-30. Mu.L of pre-chilled DEPC water.
② RNA concentration determination the concentration of extracted RNA was determined using Nanodrop 2000 software and experimental recordings were made and samples were stored at-80℃for later use or immediately for subsequent processing.
③ Reverse transcription of mRNA:
mRNA was reverse transcribed into cDNA using HISCRIPT III ALL-in-one RT SuperMix Perfect for qPCR Kit (purchased from Nanjinozan organism) and the reaction system was prepared as shown in Table 1 below.
TABLE 1mRNA reverse transcription reaction sample addition system
After the above systems are evenly mixed, the mixture is centrifuged instantaneously, and the reaction program is that the temperature is 50 ℃ for 15min and the temperature is 85 ℃ for 5s.
④qRT-PCR
Primer sequences of the related genes were designed (see Table 2), and the reaction system was prepared according to Taq Pro Universal SYBR QPCR MASTER Mix Kit (purchased from Nanjinouzan organism) instructions (see Table 3) and amplified using a real-time fluorescent quantitative PCR apparatus Q6.
TABLE 2 primer sequences for related genes
TABLE 3 mRNAqRT-sample addition System for PCR reaction
After the above systems are uniformly mixed, the mixture is subjected to instantaneous centrifugation, and the reaction program is that the mixture is subjected to pre-denaturation at 95 ℃ for 30s, denaturation at 95 ℃ for 5s and annealing at 60 ℃ for 34s, data are acquired, and after the circulation is finished, melting curve analysis is carried out. Melting curve acquisition procedure 95℃15s,60℃1min,95℃30s,60℃15s, melting curve data were acquired.
⑤ Analysis of experimental results
After the reaction, confirming an amplification curve and a melting curve of qRT-PCR, collecting Ct values of each template, calculating by using a2-△△Ct method, and correcting mRNA by using GAPDH as an internal reference.
Example 2circ96498 inhibits proliferation of irradiated hepatic stellate cells
SiRNA si-circ96498 or control si-circNC (both purchased from Oryza guangzhou Ruibo) transfected circ96498 in hepatic stellate cells LX2 were irradiated for 24h with 8Gy and then tested for the cell proliferation capacity of irradiated hepatic stellate cells using CCK-8 and EdU experiments. The results show that si-circ96498 significantly enhanced the cell proliferation capacity of irradiated hepatic stellate cells (A, C in fig. 2). Meanwhile, the overexpressing plasmid circ96498-OE or control circNC-OE (both purchased from Gicestode Guangzhou) of circ96498 was transfected in hepatic stellate cells LX2, irradiated with 8Gy after 24h, and then tested for the cell proliferation capacity of irradiated hepatic stellate cells using CCK-8 and EdU experiments. The results show that circ96498-OE significantly inhibited the cell proliferation capacity of irradiated hepatic stellate cells (FIG. 2). In conclusion, up-regulating expression circ96498 can obviously inhibit proliferation of radial hepatic stellate cells.
The detection method of the CCK-8 and EdU experiments is as follows:
(1)CCK-8
① Hepatic stellate cells were seeded in 96-well plates (3000 cells/well) and given the corresponding treatments;
② Cell proliferation activity was measured using CCK-8 kit (purchased from japan homozygote) at 6, 24, 48, 72, 96, 120 hours after cell irradiation, respectively;
③ According to the set time point, the medium in the wells was aspirated, washed 1 time with PBS, and then 100. Mu.L of freshly prepared solution (containing 10. Mu.L of CCK-8 reagent and 90. Mu. LDMEM medium) was added to each well and incubation was continued for 2h;
④ The absorbance at 450nm was read using an microplate reader and a cell proliferation curve was drawn.
(2)EdU
Cell proliferation activity was measured using the EdU assay kit (purchased from Ruibo organism, guangzhou).
① Plating, namely inoculating hepatic stellate cells into a 96-well plate (3000 cells/well), and giving corresponding treatment;
② EdU labeling, a proper amount of 50 mu M EdU culture medium is prepared according to the volume ratio of the complete culture medium to the EdU reagent of 1000:1. The culture medium was discarded 48h after the cell irradiation, washed once with PBS, and 100. Mu.L of 50. Mu.M EdU medium was added to each well and incubated at 37℃for 2h. The medium was discarded and the PBS was shaken 2 times (80 rpm/min) for 5min each;
③ Fixing, namely adding 50 mu L of 4% paraformaldehyde into each hole, and fixing for 30min at room temperature;
④ Glycine treatment, namely discarding the fixing solution, adding 50 mu L of 2mg/mL glycine solution into each hole, and slowly shaking (40 rpm/min) for 5min by a shaking table;
⑤ Cell permeation by discarding glycine solution, and shaking with PBS (40 rpm/min) for 5min. mu.L of penetrant (0.5% Triton-X-100 in PBS) was added to each well and the shaker was slowly shaken (40 rpm/min) for 10min. Discarding the penetrant, and shaking (80 rpm/min) with PBS for 5min;
⑥ Apollo staining, namely adding 100 mu L of 1xApollo staining solution into each hole, and keeping away light, room temperature and shaking slowly (40 rpm/min) for 30min. Discarding the staining solution, and shaking and washing with PBS (80 rpm/min) for 3 times, each time for 5min;
⑦ DNA staining, namely adding 1x hoechst33342 reaction solution into each hole, and keeping out light, at room temperature and slowly shaking by a shaking table (40 rpm/min) for 30min. Discarding the staining solution, and shaking and washing with PBS (80 rpm/min) for 3 times, each time for 5min;
⑧ And (3) microscopic examination and photographing, namely observing and collecting images under a Nikon inverted fluorescence microscope.
Example 3circ96498 promotes apoptosis of irradiated hepatic stellate cells
The apoptosis of irradiated hepatic stellate cells was detected by flow cytometry after 8Gy irradiation following either siRNA si-circ96498 or control si-circNC of circ96498 transfected in hepatic stellate cells LX2 according to example 2, 24 h. The results show that si-circ96498 significantly inhibited apoptosis of irradiated hepatic stellate cells (a in fig. 3). The overexpression plasmid circ96498-OE of circ96498 or control circNC-OE was transfected into hepatic stellate cells LX2, and after 24h 8Gy irradiation was performed, and after 48h apoptosis of irradiated hepatic stellate cells was detected by flow cytometry. The results show that circ96498-OE significantly enhanced apoptosis of irradiated hepatic stellate cells (FIG. 3B). In conclusion, up-regulating expression circ96498 can obviously promote apoptosis of radial hepatic stellate cells.
The detection method for detecting apoptosis by the flow cytometry comprises the following steps:
the detection was performed using an apoptosis detection kit (purchased from Jiangsu Kaiyi organisms).
① The hepatic stellate cells were inoculated in 6-well plates and given corresponding treatments;
② After 48h of cell irradiation, the supernatant was discarded, washed once with PBS, cells digested with pancreatin and collected in 1.5mL EP tubes;
③ Discarding the supernatant, and adding 500 mu L of Binding Buffer suspension cells;
④ Adding 5 mu LAnnexin V-APC, mixing, adding 5 mu LPropidium Iodide, mixing;
⑤ Reacting for 10min at room temperature in dark;
⑥ And (5) performing flow type on-machine detection within 1 h.
Example 4circ96498 inhibits secretion of radial hepatic stellate cell inflammatory factor
SiRNA si-circ96498 or control si-circNC to circ96498 was transfected into hepatic stellate cells LX2 according to example 2, 8Gy irradiated after 24h, and the culture supernatants of the cells were collected after 48h irradiation and assayed for expression of inflammatory factors TNF- α, IL-6 and IL-1β by ELISA. The results show that si-circ96498 significantly promoted secretion of the inflammatory factors TNF- α, IL-6 and IL-1β from irradiated hepatic stellate cells (FIG. 4A). The overexpressing plasmid circ96498-OE of circ96498 or control circNC-OE was transfected in hepatic stellate cells LX2, irradiated with 8Gy after 24h, and the cell culture supernatants were collected after 48h irradiation and assayed for expression of the inflammatory factors TNF- α, IL-6 and IL-1β by ELISA. The results show that circ96498-OE significantly inhibited secretion of the inflammatory factors TNF- α, IL-6 and IL-1β from irradiated hepatic stellate cells (FIG. 4B). In conclusion, overexpression of circ96498 significantly inhibited secretion of the inflammatory factors TNF- α, IL-6 and IL-1β from irradiated hepatic stellate cells.
The detection method of the ELISA is as follows:
the levels of TNF- α, IL-6 and IL-1. Beta. In the cell supernatants were measured using the TNF- α, IL-6 and IL-1. Beta. Cytokine ELISA detection kit (purchased from Shanghai ELISA). The method comprises the following specific steps:
① Collecting the sample, namely collecting cell supernatant, centrifuging at 2000rpm at room temperature for 10min, and sucking the supernatant to collect in a 1.5mL centrifuge tube;
② Standard sample adding, namely setting a standard sample hole and a sample hole, wherein 50 mu L of standard samples with different concentrations are added into each standard sample hole;
③ And (3) sample adding, namely respectively arranging blank holes (a blank control hole is not added with a sample and an enzyme-labeled reagent, and the rest steps are the same), and a sample hole to be tested. Firstly adding 40 mu L of sample diluent into a sample hole to be detected on an enzyme-labeled coating plate, and then adding 10 mu L of sample to be detected (the final dilution of the sample is 5 times);
④ Adding enzyme, namely adding 100 mu L of enzyme-labeled reagent into each hole except for blank holes;
⑤ Incubation, namely sealing the plate by using a sealing plate membrane and then incubating for 1h at 37 ℃;
⑥ Preparing a liquid, namely diluting the 20-time concentrated washing liquid with distilled water for later use;
⑦ Carefully removing the sealing plate film, discarding the liquid, spin-drying, filling the washing agent into each hole, standing for 30s, discarding, and repeating for 5 times;
⑧ Color development, namely adding 50 mu L of color development liquid A into each hole, adding 50 mu L of color development liquid B, gently shaking and uniformly mixing, and developing for 15min at 37 ℃ in a dark place;
⑨ Measuring absorbance (OD value) of each well sequentially at a wavelength of Kong Diaoling and 450 nm;
⑩ And (3) calculating, namely collecting data, drawing a standard curve of each cytokine, generating a corresponding formula, and calculating the content of each cytokine.
Example 5circ96498 inhibits the fibrotic phenotype of irradiated hepatic stellate cells
SiRNA si-circ96498 or control si-circNC of circ96498 was transfected into hepatic stellate cells LX2 according to example 2, and after 24h 8Gy irradiation was performed, after 48h irradiation, the expression of the irradiated hepatic stellate cells α -SMA and collagen1 proteins was detected using Westernblot and immunofluorescence. The results show that si-circ96498 significantly promoted the expression of the irradiated hepatic stellate cell fibrosis factor α -SMA and collagen1 proteins (A, B in fig. 5). The expression of the hepatic stellate cell alpha-SMA and collagen1 proteins was detected by Westernblot or immunofluorescence after 8Gy irradiation was performed 24h after transfection of the overexpressing plasmid circ96498-OE of circ96498 or control circNC-OE in hepatic stellate cell LX2, followed by 48h irradiation. The results show that overexpression of circ96498-OE significantly inhibited the expression of the irradiated hepatic stellate cell fibrosis factor α -SMA and collagen1 proteins (A, B in FIG. 5).
The detection method of Western blot and immunofluorescence is as follows:
(1)Westernblot
① Cell lysis, namely adding a proper amount of RIPA protein lysate into cells to be detected to fully lyse the cells.
② Protein concentration determination and sample preparation, namely detecting the protein concentration by using a BCA method, blowing and mixing uniformly according to the proportion of the protein volume of 5×loading buffer=4:1, and heating the mixed sample in a metal bath at 100 ℃ for 5min.
③ Electrophoresis, namely preparing concentrated gel of 10% SDS-PAGE and 5% SDS-PAGE, sampling 20 mug protein sample, setting electrophoresis voltage, concentrating gel (rubberizing) for 60V, separating gel (rubberizing) for 90V, and switching off power supply when bromophenol blue reaches the bottom of the separating gel rapidly, wherein the electrophoresis time is 60V,20min, and the gel separating (gumming) time is 90V,80-90 min.
④ Transferring membrane, namely after electrophoresis, setting constant current 200mA for 2h and transferring protein to PVDF membrane under low temperature condition (4 ℃).
⑤ Blocking PVDF membrane was blocked by immersing in 5% BSA and shaking on a slow (40 rpm/min) shaker at room temperature for 1h.
⑥ After blocking, the PVDF membrane is taken out, immersed in TBST (1% TBST=1L TBS buffer+1mL Tween-20) solution for 5min, and then placed in the prepared primary antibody solution, and incubated overnight on a 4 ℃ slow shaker.
⑦ The next day, PVDF membrane is taken out, placed on a rapid (120 r/min) shaking table at room temperature, washed with TBST solution for 10min each time, and repeated 3 times. The washed PVDF membrane was placed in the corresponding secondary antibody solution and shaken on a slow (40 rpm/min) shaker at room temperature for 1h.
⑧ And (3) after the secondary antibody is incubated, washing the PVDF film for 3 times by using TBST solution, and collecting a strip image by using ECL chemiluminescence.
(2) Immunofluorescence
① Plating, namely inoculating LX2 cells into a 24-well plate, performing intervention treatment, removing a culture medium, and washing with PBS for 1 time;
② Fixing by adding 0.5mL of 4% paraformaldehyde fixing solution, and fixing for 30min;
③ Washing, namely discarding the fixed liquid, washing for 3 times by using PBS, and slowly shaking (40 rpm/min) by using a shaking table for 5min each time;
④ Blocking, namely adding 0.5mL of 5% BSA for blocking, and standing for 1h at room temperature;
⑤ Applying primary antibody, namely discarding the sealing solution, adding 0.2mL of the primary antibody diluted in proportion, and slowly shaking (40 rpm/min) on a 4 ℃ shaking table for overnight;
⑥ Applying secondary antibody, discarding primary antibody, washing with PBS for 3 times, shaking slowly (40 rpm/min) for 5min each time, adding 0.2mL of the secondary antibody diluted in proportion, and shaking slowly (40 rpm/min) at room temperature in dark place for 1 hr;
⑦ The method comprises the steps of (1) nuclear staining, secondary antibody recovery, PBS washing for 3 times, shaking table slowly (40 rpm/min) for 5min each time, adding 0.2mL nuclear staining solution (DAPI), and shaking table slowly (40 rpm/min) for 5min at room temperature in dark place;
⑧ Washing, namely discarding the cell nucleus staining solution, washing for 3 times by using PBS, and slowly shaking (40 rpm/min) by using a shaking table for 5min each time;
⑨ Photographing, observing and taking a fluorescence photograph by using an inverted fluorescence microscope IX 73.
Example 6circ96498 inhibition of NF- κB and JNK/Smad2 signalling pathways radiating hepatic stellate cells
SiRNA si-circ96498 or control si-circNC of circ96498 was transfected into hepatic stellate cells LX2 according to example 2, and after 24h 8Gy irradiation was performed, and after 48h irradiation, western blot was used to detect expression of the molecules involved in the NF- κB and JNK/Smad2 signaling pathways of irradiated hepatic stellate cells. The results show that si-circ96498 was able to significantly activate NF- κB and JNK/Smad2 signaling pathways radiating hepatic stellate cells (FIG. 6A). The expression of the relevant molecules of the signal paths of the irradiated hepatic stellate cells NF- κB and JNK/Smad2 is detected by Western blot after 8Gy irradiation is carried out after the overexpression plasmid circ96498-OE of circ96498 or control circNC-OE is transfected in the hepatic stellate cells LX2 for 24 hours. The results show that circ96498-OE significantly inhibited NF-. Kappa.B and JNK/Smad2 signaling pathways in irradiated hepatic stellate cells (FIG. 6B).
Example 7 in vivo experiments demonstrated that circ96498 inhibited radioactive liver fibrosis in mice
Female C57BL/6 mice from 6-8 weeks were randomized and divided into non-radiotherapy (NIR, n=6), radiotherapy (ir+vector, n=6) and radiotherapy+knock down circ96498 (ir+sh-circ 96498, n=6). One week prior to radiotherapy, mice were given their tail intravenous AAV-Gfap-shcirc96498 (purchased from han-Hemsl) to specifically knock out the expression of mouse hepatic stellate cell circ96498, or to inject a blank vector (vector) as a control. The right liver lobes (8 gy×5, once a day) of the mice were irradiated with X-rays to construct a radioactive liver fibrosis model. Mice were sacrificed 4 weeks after the last irradiation, and the right liver tissue and serum of the mice were collected to evaluate the condition of radioactive liver fibrosis of the mice.
Right liver tissue of mice was ground and tissue RNA was extracted, and qRT-PCR was used to detect the expression of inflammatory factors IL1b, IL6 and Tnf- α mRNA, which indicated that radiotherapy upregulated the expression of inflammatory factors IL1b, IL6 and Tnf- α mRNA, while knocking down circ96498 in hepatic stellate cells further upregulated the expression of inflammatory factors IL1b, IL6 and Tnf- α mRNA (FIG. 7A). The qRT-PCR detection method described above is described in example 1, wherein the sequences of the primers used are as follows in Table 4:
TABLE 4 primer sequences for related genes
Mouse serum was taken and assayed for the expression of the inflammatory factors IL-1β, IL-6 and TNF- α using ELISA, which showed that radiotherapy upregulated the expression of the inflammatory factors IL-1β, IL-6 and TNF- α, whereas knockdown of circ96498 in hepatic stellate cells further upregulated the expression of the inflammatory factors IL-1β, IL-6 and TNF- α (FIG. 7B). In summary, knocking down hepatic stellate cell circ96498 expression in vivo promotes the formation of liver inflammatory environment, exacerbating liver inflammation.
The liver fibrosis of the mice was assessed by taking the right liver tissue line HE, immunohistochemistry and sirius red staining of the mice. The results showed that radiotherapy promoted expression of mouse liver α -SMA, aggravated its liver fibrosis, whereas knocking down circ96498 in hepatic stellate cells further up-regulated expression of α -SMA, further aggravated fibrosis (A, B in fig. 8). In addition, ELISA examined the expression of mouse serum ALT and AST, and found that radiotherapy up-regulated the expression level of mouse serum ALT and AST, resulting in liver injury, while knocking down circ96498 in hepatic stellate cells further increased the expression level of serum ALT and AST, further aggravated the liver injury (FIG. 8B).
The above-mentioned immunohistochemical detection method is as follows:
① Slicing and baking, namely fixing the collected specimens for 48 hours by using 4% paraformaldehyde, and dehydrating tissues and embedding paraffin. Slicing, and placing the slices in a 65 ℃ oven for 2 hours before an experiment;
② Dewaxing and alcohol gradient dehydration, namely, xylene I10min, xylene II 10min, xylene III 10min, 100% ethanol I5 min, 100% ethanol II 5min, 90% ethanol 5min, 80% ethanol 5min, 70% ethanol 5min and PBS soaking for 5min;
③ High pressure antigen retrieval, namely pouring the prepared 1 Xcitric acid buffer solution into a box, overflowing the box through a glass slide, putting the box into a pressure cooker, and adding a small amount of citric acid buffer solution into the pressure cooker. When the water is boiled, the cover is covered, and after the air is sprayed for 5min, the heating is stopped. Taking out the wafer box and cooling at room temperature;
④ Sealing, namely treating the tissue of the slide with 3% hydrogen peroxide before sealing, dripping the hydrogen peroxide on the tissue, incubating for 15min, and then washing the slide with PBS slowly for 5min each time for 3 times. Gently spin-drying the slide, and dripping goat serum for incubation and slicing for 30min;
⑤ Incubation of primary antibody, namely lightly spin-drying a slide, dripping a proper volume of primary antibody, and incubating at 4 ℃ overnight;
⑥ The second antibody is incubated, and the temperature of the slide glass is rewarmed for 30min at room temperature on the next day. The mixture was shaken 3 times with PBS at 100rpm/min for 5min each time. Adding secondary antibody with proper volume, incubating for 1h at room temperature, and then shaking and washing with PBS for 3 times;
⑦ DAB developing, namely, gently spin-drying the slide, immediately timing and observing the dyeing reaction condition of the tissue under a mirror after dropping the DAB developing agent on the tissue, timely putting the slide into PBS according to the dyeing condition to stop developing, recording the dyeing time, and uniformly dyeing the slide which needs to be subjected to dyeing intensity contrast;
⑧ Counterstaining with hematoxylin and returning blue, namely placing the developed flakes in a basin, washing with running water for 5min, immersing in hematoxylin for 2min, washing with running water for 2min, differentiating with 1% hydrochloric acid alcohol for 6s, and washing with running water for 10min;
⑨ Dehydrating and sealing the tablet, namely 75% ethanol for 5min, 85% ethanol for 5min, 95% ethanol for 5min, 100% ethanol for 5min, dimethylbenzene for 5min and dimethylbenzene for 5min. Naturally airing, and sealing the sheet by using neutral resin.
⑩ Microscopic examination, observing with a positive microscope and photographing.
In conclusion, knocking down circ96498 in hepatic stellate cells promotes radioactive liver injury and radioactive liver fibrosis in mice. Consistent with the experimental result of the cell level, it is proved that the circ96498 can negatively regulate the progress of the radioactive liver injury and the radioactive liver fibrosis diseases, and can prevent the progress of the radioactive liver injury and the radioactive liver fibrosis diseases by up-regulating the expression level of the circ96498, thereby providing a new important target and a new path for preventing and treating the radioactive liver injury and the radioactive liver fibrosis diseases.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.